Approach To The Destructive Effect Of Osteoclast On Cartilage And Its Differentiation Factors In Synovial Tissue Of Ankylosing Spondylitis

The chief pathological manifestations of ankylosing spondylitis (AS) includes paraplasm of synovial tissue in affected joint, massive monocyte and polykaryocytes infiltration, formation of ocular pannus with erosion on bone and cartilage surface, focal osteoclasia on adjacent spots; ossification rigidity of ligaments, synovial tissue and intervertebral disks and systemic or focal osteoporosis could be observed in later stage patients. Osteoporosis and cartilage osteoclasia are commonly observed in inflammatory arthropathy and multiple rheumatoid diseases, research on animal model of arthritis and rheumatoid arthritis have confirmed that osteoclast (OC) had a increased number and activity during the pathogenesis, which played a critical role in osteoporosis and osteoclasia. Numerous researches confirmed that OC and its precursor derives from mononuclear macrophages in hematopoietic system, active OC can originate from multiple sources including mononuclear macrophages in peripheral blood, pulmonary alveolar macrophages, spleen cell and alveolar macrophages etc. The effect of osteoblast/stroma cells are indispensable to the process of precursor differentiate into mature OC, which regulated the maturation, development and differentiation of OC. Without the involvement and contact with OB/SC, neither precursors could not differentiate into matured OC, nor matured would activate into functional OC. Approach to the differentiation of OC from molecular perspective became a hot-spot, osteprotegerin (OPG)/ receptor activator of nuclear factorκB ligand, (RANKL)/ receptor activator of nuclear factorκB (RANK), colony stimulating factor of macrophages(M-CSF), vascular endothelial cell growth factor (VEGF), tumor necrosis factor-α(TNF-α)have been found to related with the promotion of OC differentiation and maturation. Bone matrix is degraded by acid, lysosomal enzyme and protease secretion from matured OC, among which matrix metalloproteinase-3 (MMP-3) is critical to the degradation of cartilage matrix, and its expression have been found on peripheral blood and synovial fluid of AS patients. The massive mononuclear macrophages on AS synovial tissue constituted the hyperplasia of synovial tissue, whether theses cells can be identified as osteoclast precursor, do they involved the degradation of focal cartilage destruction, if the mechanism is related with MMP-3, or any differentiation related factors existed in synovial tissue still requires further investigation.Objective: the research utilized synovial tissue from sacroiliac and iliac joints of AS patients, approach to the routes and effect of OC on cartilage damage through cultivate OC and analyze its relationship; and approach to the factors and routes of OC differentiation through observation the expression of differentiation factor TNF-αand VEGF and OB in synovial tissue of AS.Method: synovial tissue and cartilage separated from sacroiliac joint of AS patients were prepared into paraffin and frozen section, cartilage pathological changes were observed through HE and toluidine blue stain, and mankin score was made; the distribution of TRAP+ cells as well as the positive areas on TRAP enzyme histochemical staining were recorded, and its relationship with Mankin score were inspected by Spearman correlation analysis; the expression of MMP-3 mRNA in synovial tissue were conducted by in situ hybridization, and the grayscale difference with normal tissue were compared by a image analysis system; expression of MMP on TRAP+ cells and the relationship between TRAP+ cells and MMP-3 were analyzed through enzyme histochemistry combined with in sity hybridization; the separated mononuclear macrophage in synovial tissue of AS were cultivated with normal joint cartilage in vitro, osteoclasia was inspected by HE staining and electron microscope, which further investigated the effect and route of cartilage damage from TRAP+ cells. Expression of mRNA of TNF-αand VEGF in synovial tissue were inspected by in situ hybridizatin, and the grayscale difference with normal tissue were compared by a image analysis system; a Spearman correlation analysis was conducted to determine its relationship with TRAP+ area and MMP-3mRNA; synovial fibroblast were separated and cultivated in vitro, the 3rd generation were collected for osteoblast identification: alkaline phosphatase by calcium-cobalt method, type I collagen by immunohistochemistry, calcium nodule by alizarin red staining and amount of osteocalcin by radio-immunity.Results: 1.part cells in synovial lining and synovial-cartilage border became red on enzyme-histochemistry-TRAP staining, with negative results in control group, the difference between staining areas in two positive groups was of statistical significance (P<0.01); caritilage damage were inspected by HE and toluidine blue staining, and histopathology Mankin score were made, the score was significantly correlate with TRAP positive area (r=0.712,P=0.001). 2. MMP-3 mRNA expression by in situ hybridization observed brown particles in synovial lining and inferior cells, with negative results in control group, the grayscale comparison returned a statistical significant difference (P<0.01); the grayscale of MMP-3 mRNA positive cells, TRAP positive areas and Mankin score were correlated (r=0.491,P=0.024 ; r=0.728,P=0.001), in situ hybridization combined with enzyme histochemistry observed that brown particles within cells changed into copper-brown. 3. the separation of mononuclear macrophage in synovial tissue of AS was successful, microscopy on TRAP staining found cells in big size and multiple nucleus, and red particles in plasma; the co-cultivation of macrophage and normal joint cartilage discovered irregular shed on cartilage surface, collagen exposure and break-down in HE staining and electro microscope. 4. TNF-αand VEGF mRNA expression test by in situ hybridization observed brown particle in sinovial lining and inferior cells, with negative results in control group, the grayscale difference between two groups were of statistical significance (P<0.01), the grayscale of TNF-αmRNA positive cells correlated with TRAP positive area ratio and Mankin score (r=0.634,P=0.002;r=0.851,P=0.001), while significant correlation between gray scale of VEGF mRNA positive cells between TRAP positive area ratio and Mankin score were recored (r=0.598,P=0.004;r=0.851,P=0.001). 5. osteoblast identification of 3rd generation fibroblasts: alkaline phosphatase by calcium-cobalt revealed black particles in fibroblasts, negative in control group; while yellow particles were found in type I collagen test by immunohistochemistry, negative in control group; the amount of osteocalcin in experiment group is significantly different with control group by radio-immuno method (P<0.01); on day 18 of fibroblast cultivation, calcium nodule formation was observed by alizarin red staining.Conclusion: 1. the presence of OC in synovial tissue correlated with cartilage damage in AS, the expression of MMP-3 is a key route. 2. The synovial tissue in AS formed a environment for the differentiation of OC: osteoblast-like characterization of fibroblasts, expression of TNF and VEGF are major environmental factors for OC differentiation. 3. the differentiation of fibroblasts into osteoblasts in AS also contributed to the pathological bone formation.